This subproject is one of many research subprojects utilizing the resources provided by a Center grant funded by NIH/NCRR. Primary support for the subproject and the subproject's principal investigator may have been provided by other sources, including other NIH sources. The Total Cost listed for the subproject likely represents the estimated amount of Center infrastructure utilized by the subproject, not direct funding provided by the NCRR grant to the subproject or subproject staff. Homologous recombination (HR) is key to repairing double-strand DNA breaks. HR defects and double-strand break repair defects cause several human cancer-related syndromes, suggesting that HR and related DNA repair pathways are vital to genome integrity. We focus on determining regulation of a "network" composed of HR and downstream enzymes, by applying combining biochemical, molecular, and genetic approaches in yeast. Preliminary data suggest there are multiple HR pathways, distinguished by Sgs1, Mus81 and RNase H2. A major focus is to determine the roles of RNase H2 in recombination-mediated DNA repair. RNase H2 has a minor role in Okazaki fragment processing in DNA replication. RNase H2 mutations in humans cause Aicardi-Goutieres syndrome, a fatal neurological disease. Our previous study indicated that RNase H2 plays significant roles in DNA repair along with Sgs1 and Mus81, which function downstream of HR. We define how RNase H2 functions downstream of HR. Based on our hypothesis that RNase H2 makes a larger complex for DNA repair, native RNase H2 supercomplex will be purified, assayed for nuclease activity and substrate specificity. Another project seeks to define the in vivo function of Mus81. Our previous study identified novel functions of Mus81 in vivo, including roles in checkpoint control and rDNA expansion/contraction. We identify other functions of Mus81 in vivo, testing protein modification including SUMOylation and ubiquitylation. These experiments'results provide insights into maintenance of genome stability controlled by HR and related enzymes;and they increase understanding of maintenance of genome stability in humans.